International Journal of Systematic and Evolutionary Microbiology (2015), 65, 135–140
DOI 10.1099/ijs.0.069336-0
Mesonia aquimarina sp. nov., a marine bacterium isolated from coastal seawater Ahyoung Choi, Kiwoon Baek,3 Hansol Lee and Jang-Cheon Cho Correspondence
Department of Biological Sciences, Inha University, Incheon 402-751, Republic of Korea
Jang-Cheon Cho [email protected]
A Gram-staining-negative, aerobic, yellow, non-flagellated, non-gliding, rod-shaped bacterium, designated strain IMCC1021T, was isolated off the coast of the East Sea. Optimal growth of strain IMCC1021T was observed at 25 6C, pH 7.0–7.5 and in the presence of 3.0–3.5 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the strain belonged to the genus Mesonia, showing a close relationship with Mesonia mobilis KMM 6059T (97.4 % similarity) followed by Mesonia phycicola MDSW-25T (96.1 %). DNA–DNA relatedness between strain IMCC1021T and M. mobilis KMM 6059T was 17.5 % (reciprocal 7.4 %), which indicated that the new isolate represents a novel genomic species of the genus Mesonia. The major fatty acids were iso-C15 : 0 and iso-C17 : 0 3-OH, and the G+C content of the genomic DNA was 41.4 mol%. Strain IMCC1021T contained menaquinone-6 (MK-6) as the respiratory quinone, and polar lipids comprising phosphatidylethanolamine, two unknown aminolipids and three unknown polar lipids. On the basis of the phylogenetic distinctions and differential phenotypic characteristics, it is suggested that strain IMCC1021T (5KCTC 32320T5NBRC 109485T) should be assigned to the genus Mesonia as the type strain of a novel species, for which the name Mesonia aquimarina sp. nov. is proposed.
The genus Mesonia, which was first described by Nedashkovskaya et al., (2003), lies within the family Flavobacteriaceae (Jooste, 1985; Reichenbach, 1992; Bernardet et al., 1996, 2002) of the phylum Bacteroidetes (Garrity & Holt, 2001). At the time of writing, the genus Mesonia consists of four species with validly published names: Mesonia algae (Nedashkovskaya et al., 2003, the type species of the genus Mesonia), Mesonia mobilis (Nedashkovskaya et al., 2006), Mesonia phycicola (Kang & Lee, 2010) and Mesonia ostreae (Lee et al., 2012). The majority of these are of marine origin. Members of the genus Mesonia are characterized as being Gram-stainingnegative, flexirubin-negative, rod-shaped bacteria that contain MK-6 as the major respiratory quinone and phosphatidylethanolamine as the major polar lipid. In the present study, we describe strain IMCC1021T, isolated from a surface seawater sample from the East Sea. Based on the data from physiological, chemotaxonomic and phylogenetic analyses, it is proposed that strain IMCC1021T represents a novel species of the genus Mesonia. Strain IMCC1021T was isolated from a seawater sample collected off the coast of the East Sea (38u 299 58.640 N 3Present address: Division of Polar Life Sciences, Korea Polar Research Institute, 26 Songdomirae-ro, Yeonsu-gu, Incheon 406-840, Republic of Korea. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain IMCC1021T is KJ572118. Two supplementary figures are available with the online Supplementary Material.
069336 G 2015 IUMS
Printed in Great Britain
128u 259 35.590 E), using a standard dilution plating method on marine agar 2216 (MA; BD Difco) at 20 uC. After the optimum growth temperature had been determined, cultures of strain IMCC1021T were routinely maintained on MA or in marine broth (MB; BD Difco) at 25 uC. For physiological and chemotaxonomic comparisons between strain IMCC1021T and four type strains of species of the genus Mesonia, M. algae KCTC 12089T, M. mobilis KCTC 12708T, M. phycicola KCTC 22373T and M. ostreae KCTC 23500T were obtained from the Korean Collection for Type Cultures (KCTC) and used as reference strains. The 16S rRNA gene was amplified from a single colony and sequenced as described previously (Cho & Giovannoni, 2003). The resultant almost-complete 16S rRNA gene sequence (1450 nt) of strain IMCC1021T was compared with the sequences held in GenBank by BLASTN (Altschul et al., 1997) and also analysed using the EzTaxon-e server (Kim et al., 2012). For phylogenetic analyses, the 16S rRNA gene sequence of strain IMCC1021T was aligned using the SILVA Incremental Aligner (SINA v.1.2.11) (http://www.arb-silva.de/ aligner/) (Pruesse et al., 2012) and imported in the ARB software package (Ludwig et al., 2004). Phylogenetic trees were generated by using neighbour-joining (Saitou & Nei, 1987) [with the Jukes–Cantor distance (Jukes & Cantor, 1969)], maximum-parsimony (Fitch, 1971) and maximumlikelihood (Felsenstein, 1981) algorithms that were programmed in MEGA 6.0 (Tamura et al., 2013). The robustness of the phylogenetic trees was confirmed by bootstrap analyses based on 1000 random replicates. Comparative 16S rRNA 135
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gene sequence analyses showed that strain IMCC1021T was related most closely to M. mobilis KMM 6059T (97.4 % similarity), followed by M. phycicola MDSW-25T (96.1 %), M. algae KMM 3909T (95.8 %) and M. ostreae T-y2T (94.9 %). In all the phylogenetic trees generated in this study, strain IMCC1021T and the four species of the genus Mesonia constituted a robust clade, which was well separated from other genera in the family Flavobacteriaceae, confirming that strain IMCC1021T belongs to the genus Mesonia (Fig. 1). As the 16S rRNA gene sequence similarity between IMCC1021T and M. mobilis KMM 6059T (97.4 %) exceeded the recommended threshold (97 %) for the determination of prokaryotic species (Stackebrandt & Goebel, 1994), genomic DNA relatedness was measured by DNA–DNA hybridization (DDH) assays. DDH was performed by membranebased slot-blot hybridization using a DIG-High Prime DNA Labelling and Detection Starter kit II (Roche Molecular Biochemicals). DNA–DNA relatedness was calculated from the relative signal intensities of three slot-blots estimated by densitometric analysis (LAS-3000, Fujifilm). The level of DNA–DNA relatedness between the two strains was 17.5 % (with IMCC1021T as the probe) and 7.4 % (with M. mobilis KCTC 12708T as the probe), which was well below the 70 % recommended for the demarcation of bacterial genomic species (Wayne et al., 1987), demonstrating that strain IMCC1021T is a novel strain representing a separate genomic species of the genus Mesonia. To characterize strain IMCC1021T phenotypically, the strain was routinely grown on MA or in MB at 25 uC for 3 days. The cellular morphology and cell size were examined by both transmission electron microscopy (TEM; CM200,
0.01
73 81*
Salegentibacter mishustinae KMM 6049T (AY576653) Salegentibacter salarius ISL-6T (EF486353) Salegentibacter holothuriorum KMM 3524T (AB116148)
100 100, 100
Salegentibacter salegens DSM 5424T (M92279) 93
81 70, 76
Philips) and phase-contrast microscopy (80i, Nikon). For TEM, cells were negatively stained with 2.0 % (w/v) uranyl acetate on a carbon-coated copper grid. The gliding motility of strain IMCC1021T was examined by phase-contrast microscopy of a hanging drop preparation (Bernardet et al., 2002). The ability of the strain to grow anaerobically was tested using the MGC anaerobic system (Mitsubishi Gas Chemical) with cultures incubated on MA at 25 uC for 2 weeks. Growth at 4, 10, 15, 20, 25, 30, 37 and 42 uC and at pH 5.0–10.0 (at 0.5 pH unit intervals) was monitored in artificial seawater medium (ASW; Choo et al., 2007) amended with 0.5 % (w/v) peptone and 0.1 % (w/v) yeast extract. The pH was adjusted using the following buffering systems: MES pH 5.0–6.0, MOPS pH 6.5–7.0, HEPES pH 7.5–8.0, Tris pH 8.5–9.0 and CHES pH 9.5–10.0. Growth with 0–5 % (w/v) NaCl (at intervals of 0.5 %) and 5.0–15.0 % [(w/v), at intervals of 2.5 %] was determined in NaCl-free ASW amended with 0.5 % (w/v) peptone and 0.1 % (w/v) yeast extract. The increase in turbidity of each culture was monitored by spectrophotometry (Optizen 2120UV, Mechasis). Gram staining was performed using a Gram-staining kit (bioMe´rieux). Hydrolysis of macromolecules was tested by incubating cultures at 25 uC on MA containing various macromolecules, and was determined based on the formation of clear zones around colonies after applying suitable staining solutions (Tindall et al., 2007). The following macromolecules were tested: gelatin (0.4 %, w/v), starch (0.2 %, w/v), CM-cellulose (0.2 %, w/v), colloidal chitin (0.5 %, w/v), casein (10 % skimmed milk, w/v) and elastin (0.5 %, w/v). Tests for the hydrolysis of Tween 20 (1.0 %, v/v) and Tween 80 (1.0 %, v/v) were also performed on MA. Degradation of DNA was evaluated
Salegentibacter agarivorans KMM 7019T (DQ191176) Salegentibacter salinarum ISL-4T (EF612764)
Mesonia mobilis KMM 6059T (DQ367409)
98
Mesonia phycicola MDSW-25T (FM882228)
98 87, 97
Mesonia aquimarina IMCC1021T (KJ572118) 100 100, 100
Mesonia algae KMM 3909T (AF536383) Mesonia ostreae T-y2T (HM234095) Zunongwangia profunda SM-A87T (CP001650) Psychroflexus torquis ACAM 623T (U85881)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, showing the relationship between strain IMCC1021T and other representatives of the family Flavobacteriaceae. Bootstrap values (.70 %) based on 1000 resamplings are shown above the nodes for the neighbour-joining method and below the nodes for the maximum-parsimony and maximumlikelihood methods, respectively. Filled circles indicate that the corresponding nodes were recovered by all three treeing methods. Open circles indicate that the corresponding node was recovered by the neighbour-joining and maximum-likelihood methods. *Bootstrap value obtained from the maximum-likelihood method. Bar, 0.01 substitutions per nucleotide position. 136
International Journal of Systematic and Evolutionary Microbiology 65
Mesonia aquimarina sp. nov.
using DNase test agar (BD Difco) supplemented with 2.0 % (w/v) NaCl. Production of H2S was investigated using triplesugar iron agar (BD Difco) amended with 2.0 % (w/v) NaCl. Other biochemical tests were performed using API 20NE, API ZYM and API 50CH test strips (bioMe´rieux) according to the manufacturer’s instructions, except for inoculating the bacterial cells into ASW medium. Susceptibility to antimicrobial agents was analysed by the disc-diffusion method using the following antibiotic discs (Oxoid; mg per disc): ampicillin (10), chloramphenicol (30), erythromycin (15), gentamicin (10), kanamycin (30), nalidixic acid (30), rifampicin (30), tetracycline (30) and vancomycin (30).
Zones of inhibition were measured for each disc, and bacterial cells were considered as resistant (inhibition zone ,10 mm) or susceptible (inhibition zone .10 mm) using the same standard as Baek et al. (2014). For API 20NE, API ZYM, API 50CH and antibiotic susceptibility tests, four type strains of species of the genus Mesonia were used as reference strains under the same culture conditions. The phenotypic characteristics of strain IMCC1021T are summarized in the species description and Table 1. Cells of strain IMCC1021T were Gram-staining-negative, aerobic, non-motile and rodshaped (Fig. S1, available in the online Supplementary Material). Table 1 shows the differential characteristics
Table 1. Phenotypic characteristics that differentiate IMCC1021T from species of the genus Mesonia Strains: 1, IMCC1021T; 2, M. algae KCTC 12089T; 3, M. mobilis KCTC 12708T; 4, M. phycicola KCTC 22373T; 5, M. ostreae KCTC 23500T. All data are from this study unless indicated otherwise. All strains were positive for the following characteristics: catalase, oxidase, alkaline phosphatase, esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase enzyme activities; hydrolysis of gelatin and Tween 20; acid production from glycerol, D-galactose, L-rhamnose, dulcitol, inositol and aesculin ferric citrate; susceptibility to erythromycin, rifampicin, tetracycline and vancomycin. All strains were negative for the following characteristics: lipase (C14), cystine arylamidase, trypsin, a-chymotrypsin, agalactosidase, b-galactosidase, b-glucuronidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase enzyme activities; flexirubin-type pigments; H2S production; hydrolysis of starch, colloidal chitin and elastin; acid production from D-arabinose, D-ribose, D-xylose, L-xylose, D-adonitol, methyl b-Dxylopyranoside, methyl a-D-mannopyranoside, methyl a-D-glucopyranoside, N-acetylglucosamine, amygdalin, salicin, cellobiose, sucrose, inulin, melezitose, raffinose, glycogen, xylitol, D-lyxose, D-fucose, D-arabitol and potassium gluconate. +, Positive; 2, negative. Characteristic
1
2
3
4
5
Gliding motility* Hydrolysis of: Tween 80 Casein CM-cellulose DNA In API 20NE Arginine dihydrolase, urease Aesculin hydrolysis Enzyme activities (API ZYM) Esterase (C4) b-Glucosidase a-Glucosidase Acid production from (API50CH): Melibiose L-Sorbose D-Sorbitol D-Mannitol, D-turanose Arbutin, L-arabitol Lactose, L-fucose Potassium 5-ketogluconate Starch D-Glucose, D-mannose, maltose L-Arabinose Erythritol D-Tagatose, potassium 2-ketogluconate D-Fructose, trehalose, gentiobiose Antibiotic susceptibility Ampicillin Chloramphenicol DNA G+C content (mol%)*
2
2
+
2
2
+ + + 2
+ + 2 2
+ 2 2 2
+ + 2 +
2 2 2 2
2 2
+ 2
2 2
2 +
2 2
+ + 2
2 2 2
2 2 +
2 + +
+ 2 2
+ + + 2 + 2 2 2 2 2 + 2 2
+ + + + 2 + + + 2 2 2 + 2
+ + + 2 2 + 2 2 + + 2 2 2
+ 2 2 + 2 2 + 2 + 2 2 2 +
2 + 2 + + 2 2 + 2 + 2 2 2
+ 2 41.4
+ + 32.7–34.0
+ + 36.1
2 + 30.0
2 2 42.1
*Data for columns 2, 3, 4 and 5 were obtained from Nedashkovskaya et al. (2003, 2006), Kang & Lee, (2010) and Lee et al. (2012), respectively. http://ijs.sgmjournals.org
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between the novel strain and the type strains of species of the genus Mesonia.
Table 2. Cellular fatty acid composition of strain IMCC1021T and members of the genus Mesonia
The DNA G+C content was determined using the HPLC method (Mesbah et al., 1989) with a Discovery C18 column (5 mm, 15 cm64.6 mm; Supelco). Fatty acid methyl esters of strain IMCC1021T and four reference strains were extracted from bacterial cultures in the late exponential phase (~3 days) grown on MA at 25 uC and determined by the Sherlock Microbial Identification System (MIDI) version 6.1 with the TSBA6 database. Isoprenoid quinones were extracted by TLC according to Minnikin et al. (1984) and analysed by HPLC (Collins, 1985). Polar lipids were extracted from lyophilized bacterial biomass and separated using two-dimensional TLC followed by detection with appropriate spray reagents (Minnikin et al. 1984; Komagata & Suzuki, 1987).
Strains: 1, IMCC1021T; 2, M. algae KCTC 12089T; 3, M. mobilis KCTC 12708T; 4, M. phycicola KCTC 22373T; 5, M. ostreae KCTC 23500T. All data were obtained from this study. Fatty acids that represented ,1.0 % are not shown. ECL, equivalent chain-length; tr, trace (,1.0 %); 2, not detected.
The DNA G+C content of strain IMCC1021T was 41.4 mol%, which was within the range of DNA G+C contents reported for the genus Mesonia (32.7–42.1 mol%). The respiratory quinone detected in strain IMCC1021T was menaquinone-6 (MK-6), which is coincident with the major quinone of the family Flavobacteriaceae. The polar lipids found in strain IMCC1021T were phosphatidylethanolamine, two unknown aminolipids and three unknown lipids (Fig. S2). The major cellular fatty acid components (.10 %) of strain IMCC1021T were iso-C15 : 0 (25.1 %), isoC17 : 0 3-OH (18.0 %), C16 : 1v7c and/or C16 : 1v6c (16.6 %) and iso-C15 : 1 G (10.8 %) (Table 2), which were similar to those found in the reference strains of species of the genus Mesonia. Phylogenetic analyses showed that strain IMCC1021T formed a robust clade with species of the genus Mesonia (Fig. 1), as well as sharing similar chemotaxonomic characteristics, including DNA G+C content, quinone, polar lipids and fatty acids. This suggests that strain IMCC1021T can be assigned to this genus. The low 16S rRNA gene sequence similarities (94.9–97.4 %) between IMCC1021T and the available species of the genus Mesonia and the genomic distinctness between strain IMCC1021T and M. mobilis (less than 70 % DNA–DNA hybridization) also suggested that strain IMCC1021T represents a novel genomic species of the genus Mesonia. In addition, phenotypic characteristics, including hydrolysis of macromolecules, enzyme activities and carbon source oxidation (Table 1) differentiated strain IMCC1021T from previously recognized species of the genus Mesonia. Therefore, strain IMCC1021T should be assigned to a novel species of the genus Mesonia, for which the name Mesonia aquimarina sp. nov. is proposed.
Fatty acid (%) Straight chain C15 : 0 C16 : 0 C18 : 0 Branched iso-C15 : 0 iso-C15 : 1 G anteiso-C15 : 0 iso-C16 : 0 iso-C16 : 1 H anteiso-C17 : 1v9c Unsaturated C15 : 1v6c C17 : 1v6c C17 : 1v8c Hydroxy C15 : 0 2-OH C15 : 0 3-OH C17 : 0 2-OH iso-C15 : 0 3-OH iso-C16 : 0 3-OH iso-C17 : 0 3-OH Summed features* 3 (C16 : 1v7c and/or C16 : 1v6c) 9 (C16 : 0 10-methyl and/or iso-C17 : 1v9c) Unknown ECL 13.565
1
2
3
4
5
3.0 1.2 tr
7.3 4.3 4.3
3.6 1.4 2
4.2 tr tr
4.1 2 2
25.1 10.8 2.9 tr 2 2
20.3 7.1 7.5 2.5 1.8 1.4
33.1 9.9 3.7 tr 2 2
31.2 11.7 2.9 1.1 1.5 2
9.7 3.1 16.7 2 2 4.3
tr 1.1 2
2.8 2.4 1.6
1.1 2.0 2
2.7 tr 2
2 3.9 2
1.5 2 1.5 3.8 1.4 18.0
1.7 2 2.3 2.6 3.0 11.7
2 2 1.3 5.5 2.0 16.9
1.3 1.3 1.6 5.1 3.3 12.7
4.2 2 10.4 2 7.7 15.3
16.6
4.2
9.5
9.5
15.7
9.3
6.3
7.1
4.2
4.8
2
2.3
tr
tr
2
*Summed features represent groups of two fatty acids that cannot be separated by the MIDI system.
Gram-staining-negative, oxidase- and catalase-positive, non-motile, non-gliding and aerobic. Cells are rod-shaped
(0.5–0.6 mm61.1–2.0 mm). Colonies are 1–2 mm in diameter, circular, convex, smooth and yellow after 3 days of incubation on MA at 25 uC. Flexirubin-type pigments are not produced. Temperature range for growth is 10–37 uC (optimum, 25 uC). The pH range for growth is pH 5.5–9.5 (optimum, pH 7.0–7.5). Growth occurs at 0.5–15 % (w/v) NaCl [optimum, 3.0–3.5 % (w/v) NaCl]. Gelatin, CMcellulose, casein, Tween 20 and Tween 80 are hydrolysed, but DNA, starch, colloidal chitin and elastin are not. H2S is not produced. Positive for gelatinase activity in API 20NE, but negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, aesculin hydrolysis and b-galactosidase. Positive for the following enzyme activities (API ZYM): alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and b-glucosidase, but negative for lipase (C14), cystine
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Description of Mesonia aquimarina sp. nov. Mesonia aquimarina (a.qui.ma.ri9na. L. fem. n. aqua water; L. adj. marinus from the sea; N.L. fem. adj. aquimarina from seawater).
Mesonia aquimarina sp. nov.
arylamidase, trypsin, a-chymotrypsin, a-galactosidase, bgalactosidase, b-glucuronidase, a-glucosidase, N-acetyl-bglucosaminidase, a-mannosidase and a-fucosidase. Positive for glycerol, erythritol, D-galactose, L-sorbose, L-rhamnose, dulcitol, inositol, D-sorbitol, arbutin, aesculin ferric citrate, melibiose and L-arabitol in API 50CH, negative for Darabinose, L-arabinose, D-ribose, D-xylose, L-xylose, D-adonitol, methyl b-D-xylopyranoside, D-glucose, D-fructose, Dmannose, D-mannitol, methyl a-D-mannopyranoside, methyl a-D-glucopyranoside, N-acetylglucosamine, amygdalin, salicin, cellobiose, maltose, lactose, sucrose, trehalose, inulin, melezitose, raffinose, starch, glycogen, xylitol, gentiobiose, Dturanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, potassium gluconate, potassium 2-ketogluconate and potassium 5-ketogluconate. Susceptible to ampicillin, erythromycin, rifampicin, tetracycline and vancomycin, but resistant to chloramphenicol, gentamicin, kanamycin and nalidixic acid. The respiratory quinone detected is menaquinone 6 (MK-6). The major phospholipids are phosphatidylethanolamine, aminophosphoslipids and unknown polar lipids. The major cellular fatty acids are iso-C15 : 0, iso-C17 : 0 3-OH, C16 : 1v7c and/or C16 : 1v6c and iso-C15 : 1 G. The type strain of the species is strain IMCC1021T (5KCTC 32320T5NBRC 109485T), isolated off the coast of the East Sea, Korea. The G+C content of the genomic DNA of the type strain is 41.4 mol%.
Acknowledgements This study was supported by the project on survey of indigenous species of Korea of the National Institute of Biological Resources (NIBR), Ministry of Environment (Korea), by Mid-Career Research Program through NRF funded by the Ministry of Science, ICT and Future Planning (NRF-2013R1A2A2A01068004), and by the Inha University Research Grant.
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characterization and the principles of comparative systematics. In Methods for General and Molecular Microbiology, pp. 330–393. Edited by C. A. Reddy, T. J. Beveridge, J. A. Breznak, G. A. Marzluf, T. M. Schmidt & L. R. Snyder. Washington, DC: American Society for Microbiology.
Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. (2013).
Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & other authors (1987). International Committee on Systematic
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DOI 10.1099/ijs.0.069336-0
Mesonia aquimarina sp. nov., a marine bacterium isolated from coastal seawater Ahyoung Choi, Kiwoon Baek,3 Hansol Lee and Jang-Cheon Cho Correspondence
Department of Biological Sciences, Inha University, Incheon 402-751, Republic of Korea
Jang-Cheon Cho [email protected]
A Gram-staining-negative, aerobic, yellow, non-flagellated, non-gliding, rod-shaped bacterium, designated strain IMCC1021T, was isolated off the coast of the East Sea. Optimal growth of strain IMCC1021T was observed at 25 6C, pH 7.0–7.5 and in the presence of 3.0–3.5 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the strain belonged to the genus Mesonia, showing a close relationship with Mesonia mobilis KMM 6059T (97.4 % similarity) followed by Mesonia phycicola MDSW-25T (96.1 %). DNA–DNA relatedness between strain IMCC1021T and M. mobilis KMM 6059T was 17.5 % (reciprocal 7.4 %), which indicated that the new isolate represents a novel genomic species of the genus Mesonia. The major fatty acids were iso-C15 : 0 and iso-C17 : 0 3-OH, and the G+C content of the genomic DNA was 41.4 mol%. Strain IMCC1021T contained menaquinone-6 (MK-6) as the respiratory quinone, and polar lipids comprising phosphatidylethanolamine, two unknown aminolipids and three unknown polar lipids. On the basis of the phylogenetic distinctions and differential phenotypic characteristics, it is suggested that strain IMCC1021T (5KCTC 32320T5NBRC 109485T) should be assigned to the genus Mesonia as the type strain of a novel species, for which the name Mesonia aquimarina sp. nov. is proposed.
The genus Mesonia, which was first described by Nedashkovskaya et al., (2003), lies within the family Flavobacteriaceae (Jooste, 1985; Reichenbach, 1992; Bernardet et al., 1996, 2002) of the phylum Bacteroidetes (Garrity & Holt, 2001). At the time of writing, the genus Mesonia consists of four species with validly published names: Mesonia algae (Nedashkovskaya et al., 2003, the type species of the genus Mesonia), Mesonia mobilis (Nedashkovskaya et al., 2006), Mesonia phycicola (Kang & Lee, 2010) and Mesonia ostreae (Lee et al., 2012). The majority of these are of marine origin. Members of the genus Mesonia are characterized as being Gram-stainingnegative, flexirubin-negative, rod-shaped bacteria that contain MK-6 as the major respiratory quinone and phosphatidylethanolamine as the major polar lipid. In the present study, we describe strain IMCC1021T, isolated from a surface seawater sample from the East Sea. Based on the data from physiological, chemotaxonomic and phylogenetic analyses, it is proposed that strain IMCC1021T represents a novel species of the genus Mesonia. Strain IMCC1021T was isolated from a seawater sample collected off the coast of the East Sea (38u 299 58.640 N 3Present address: Division of Polar Life Sciences, Korea Polar Research Institute, 26 Songdomirae-ro, Yeonsu-gu, Incheon 406-840, Republic of Korea. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain IMCC1021T is KJ572118. Two supplementary figures are available with the online Supplementary Material.
069336 G 2015 IUMS
Printed in Great Britain
128u 259 35.590 E), using a standard dilution plating method on marine agar 2216 (MA; BD Difco) at 20 uC. After the optimum growth temperature had been determined, cultures of strain IMCC1021T were routinely maintained on MA or in marine broth (MB; BD Difco) at 25 uC. For physiological and chemotaxonomic comparisons between strain IMCC1021T and four type strains of species of the genus Mesonia, M. algae KCTC 12089T, M. mobilis KCTC 12708T, M. phycicola KCTC 22373T and M. ostreae KCTC 23500T were obtained from the Korean Collection for Type Cultures (KCTC) and used as reference strains. The 16S rRNA gene was amplified from a single colony and sequenced as described previously (Cho & Giovannoni, 2003). The resultant almost-complete 16S rRNA gene sequence (1450 nt) of strain IMCC1021T was compared with the sequences held in GenBank by BLASTN (Altschul et al., 1997) and also analysed using the EzTaxon-e server (Kim et al., 2012). For phylogenetic analyses, the 16S rRNA gene sequence of strain IMCC1021T was aligned using the SILVA Incremental Aligner (SINA v.1.2.11) (http://www.arb-silva.de/ aligner/) (Pruesse et al., 2012) and imported in the ARB software package (Ludwig et al., 2004). Phylogenetic trees were generated by using neighbour-joining (Saitou & Nei, 1987) [with the Jukes–Cantor distance (Jukes & Cantor, 1969)], maximum-parsimony (Fitch, 1971) and maximumlikelihood (Felsenstein, 1981) algorithms that were programmed in MEGA 6.0 (Tamura et al., 2013). The robustness of the phylogenetic trees was confirmed by bootstrap analyses based on 1000 random replicates. Comparative 16S rRNA 135
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gene sequence analyses showed that strain IMCC1021T was related most closely to M. mobilis KMM 6059T (97.4 % similarity), followed by M. phycicola MDSW-25T (96.1 %), M. algae KMM 3909T (95.8 %) and M. ostreae T-y2T (94.9 %). In all the phylogenetic trees generated in this study, strain IMCC1021T and the four species of the genus Mesonia constituted a robust clade, which was well separated from other genera in the family Flavobacteriaceae, confirming that strain IMCC1021T belongs to the genus Mesonia (Fig. 1). As the 16S rRNA gene sequence similarity between IMCC1021T and M. mobilis KMM 6059T (97.4 %) exceeded the recommended threshold (97 %) for the determination of prokaryotic species (Stackebrandt & Goebel, 1994), genomic DNA relatedness was measured by DNA–DNA hybridization (DDH) assays. DDH was performed by membranebased slot-blot hybridization using a DIG-High Prime DNA Labelling and Detection Starter kit II (Roche Molecular Biochemicals). DNA–DNA relatedness was calculated from the relative signal intensities of three slot-blots estimated by densitometric analysis (LAS-3000, Fujifilm). The level of DNA–DNA relatedness between the two strains was 17.5 % (with IMCC1021T as the probe) and 7.4 % (with M. mobilis KCTC 12708T as the probe), which was well below the 70 % recommended for the demarcation of bacterial genomic species (Wayne et al., 1987), demonstrating that strain IMCC1021T is a novel strain representing a separate genomic species of the genus Mesonia. To characterize strain IMCC1021T phenotypically, the strain was routinely grown on MA or in MB at 25 uC for 3 days. The cellular morphology and cell size were examined by both transmission electron microscopy (TEM; CM200,
0.01
73 81*
Salegentibacter mishustinae KMM 6049T (AY576653) Salegentibacter salarius ISL-6T (EF486353) Salegentibacter holothuriorum KMM 3524T (AB116148)
100 100, 100
Salegentibacter salegens DSM 5424T (M92279) 93
81 70, 76
Philips) and phase-contrast microscopy (80i, Nikon). For TEM, cells were negatively stained with 2.0 % (w/v) uranyl acetate on a carbon-coated copper grid. The gliding motility of strain IMCC1021T was examined by phase-contrast microscopy of a hanging drop preparation (Bernardet et al., 2002). The ability of the strain to grow anaerobically was tested using the MGC anaerobic system (Mitsubishi Gas Chemical) with cultures incubated on MA at 25 uC for 2 weeks. Growth at 4, 10, 15, 20, 25, 30, 37 and 42 uC and at pH 5.0–10.0 (at 0.5 pH unit intervals) was monitored in artificial seawater medium (ASW; Choo et al., 2007) amended with 0.5 % (w/v) peptone and 0.1 % (w/v) yeast extract. The pH was adjusted using the following buffering systems: MES pH 5.0–6.0, MOPS pH 6.5–7.0, HEPES pH 7.5–8.0, Tris pH 8.5–9.0 and CHES pH 9.5–10.0. Growth with 0–5 % (w/v) NaCl (at intervals of 0.5 %) and 5.0–15.0 % [(w/v), at intervals of 2.5 %] was determined in NaCl-free ASW amended with 0.5 % (w/v) peptone and 0.1 % (w/v) yeast extract. The increase in turbidity of each culture was monitored by spectrophotometry (Optizen 2120UV, Mechasis). Gram staining was performed using a Gram-staining kit (bioMe´rieux). Hydrolysis of macromolecules was tested by incubating cultures at 25 uC on MA containing various macromolecules, and was determined based on the formation of clear zones around colonies after applying suitable staining solutions (Tindall et al., 2007). The following macromolecules were tested: gelatin (0.4 %, w/v), starch (0.2 %, w/v), CM-cellulose (0.2 %, w/v), colloidal chitin (0.5 %, w/v), casein (10 % skimmed milk, w/v) and elastin (0.5 %, w/v). Tests for the hydrolysis of Tween 20 (1.0 %, v/v) and Tween 80 (1.0 %, v/v) were also performed on MA. Degradation of DNA was evaluated
Salegentibacter agarivorans KMM 7019T (DQ191176) Salegentibacter salinarum ISL-4T (EF612764)
Mesonia mobilis KMM 6059T (DQ367409)
98
Mesonia phycicola MDSW-25T (FM882228)
98 87, 97
Mesonia aquimarina IMCC1021T (KJ572118) 100 100, 100
Mesonia algae KMM 3909T (AF536383) Mesonia ostreae T-y2T (HM234095) Zunongwangia profunda SM-A87T (CP001650) Psychroflexus torquis ACAM 623T (U85881)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, showing the relationship between strain IMCC1021T and other representatives of the family Flavobacteriaceae. Bootstrap values (.70 %) based on 1000 resamplings are shown above the nodes for the neighbour-joining method and below the nodes for the maximum-parsimony and maximumlikelihood methods, respectively. Filled circles indicate that the corresponding nodes were recovered by all three treeing methods. Open circles indicate that the corresponding node was recovered by the neighbour-joining and maximum-likelihood methods. *Bootstrap value obtained from the maximum-likelihood method. Bar, 0.01 substitutions per nucleotide position. 136
International Journal of Systematic and Evolutionary Microbiology 65
Mesonia aquimarina sp. nov.
using DNase test agar (BD Difco) supplemented with 2.0 % (w/v) NaCl. Production of H2S was investigated using triplesugar iron agar (BD Difco) amended with 2.0 % (w/v) NaCl. Other biochemical tests were performed using API 20NE, API ZYM and API 50CH test strips (bioMe´rieux) according to the manufacturer’s instructions, except for inoculating the bacterial cells into ASW medium. Susceptibility to antimicrobial agents was analysed by the disc-diffusion method using the following antibiotic discs (Oxoid; mg per disc): ampicillin (10), chloramphenicol (30), erythromycin (15), gentamicin (10), kanamycin (30), nalidixic acid (30), rifampicin (30), tetracycline (30) and vancomycin (30).
Zones of inhibition were measured for each disc, and bacterial cells were considered as resistant (inhibition zone ,10 mm) or susceptible (inhibition zone .10 mm) using the same standard as Baek et al. (2014). For API 20NE, API ZYM, API 50CH and antibiotic susceptibility tests, four type strains of species of the genus Mesonia were used as reference strains under the same culture conditions. The phenotypic characteristics of strain IMCC1021T are summarized in the species description and Table 1. Cells of strain IMCC1021T were Gram-staining-negative, aerobic, non-motile and rodshaped (Fig. S1, available in the online Supplementary Material). Table 1 shows the differential characteristics
Table 1. Phenotypic characteristics that differentiate IMCC1021T from species of the genus Mesonia Strains: 1, IMCC1021T; 2, M. algae KCTC 12089T; 3, M. mobilis KCTC 12708T; 4, M. phycicola KCTC 22373T; 5, M. ostreae KCTC 23500T. All data are from this study unless indicated otherwise. All strains were positive for the following characteristics: catalase, oxidase, alkaline phosphatase, esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase enzyme activities; hydrolysis of gelatin and Tween 20; acid production from glycerol, D-galactose, L-rhamnose, dulcitol, inositol and aesculin ferric citrate; susceptibility to erythromycin, rifampicin, tetracycline and vancomycin. All strains were negative for the following characteristics: lipase (C14), cystine arylamidase, trypsin, a-chymotrypsin, agalactosidase, b-galactosidase, b-glucuronidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase enzyme activities; flexirubin-type pigments; H2S production; hydrolysis of starch, colloidal chitin and elastin; acid production from D-arabinose, D-ribose, D-xylose, L-xylose, D-adonitol, methyl b-Dxylopyranoside, methyl a-D-mannopyranoside, methyl a-D-glucopyranoside, N-acetylglucosamine, amygdalin, salicin, cellobiose, sucrose, inulin, melezitose, raffinose, glycogen, xylitol, D-lyxose, D-fucose, D-arabitol and potassium gluconate. +, Positive; 2, negative. Characteristic
1
2
3
4
5
Gliding motility* Hydrolysis of: Tween 80 Casein CM-cellulose DNA In API 20NE Arginine dihydrolase, urease Aesculin hydrolysis Enzyme activities (API ZYM) Esterase (C4) b-Glucosidase a-Glucosidase Acid production from (API50CH): Melibiose L-Sorbose D-Sorbitol D-Mannitol, D-turanose Arbutin, L-arabitol Lactose, L-fucose Potassium 5-ketogluconate Starch D-Glucose, D-mannose, maltose L-Arabinose Erythritol D-Tagatose, potassium 2-ketogluconate D-Fructose, trehalose, gentiobiose Antibiotic susceptibility Ampicillin Chloramphenicol DNA G+C content (mol%)*
2
2
+
2
2
+ + + 2
+ + 2 2
+ 2 2 2
+ + 2 +
2 2 2 2
2 2
+ 2
2 2
2 +
2 2
+ + 2
2 2 2
2 2 +
2 + +
+ 2 2
+ + + 2 + 2 2 2 2 2 + 2 2
+ + + + 2 + + + 2 2 2 + 2
+ + + 2 2 + 2 2 + + 2 2 2
+ 2 2 + 2 2 + 2 + 2 2 2 +
2 + 2 + + 2 2 + 2 + 2 2 2
+ 2 41.4
+ + 32.7–34.0
+ + 36.1
2 + 30.0
2 2 42.1
*Data for columns 2, 3, 4 and 5 were obtained from Nedashkovskaya et al. (2003, 2006), Kang & Lee, (2010) and Lee et al. (2012), respectively. http://ijs.sgmjournals.org
137
A. Choi and others
between the novel strain and the type strains of species of the genus Mesonia.
Table 2. Cellular fatty acid composition of strain IMCC1021T and members of the genus Mesonia
The DNA G+C content was determined using the HPLC method (Mesbah et al., 1989) with a Discovery C18 column (5 mm, 15 cm64.6 mm; Supelco). Fatty acid methyl esters of strain IMCC1021T and four reference strains were extracted from bacterial cultures in the late exponential phase (~3 days) grown on MA at 25 uC and determined by the Sherlock Microbial Identification System (MIDI) version 6.1 with the TSBA6 database. Isoprenoid quinones were extracted by TLC according to Minnikin et al. (1984) and analysed by HPLC (Collins, 1985). Polar lipids were extracted from lyophilized bacterial biomass and separated using two-dimensional TLC followed by detection with appropriate spray reagents (Minnikin et al. 1984; Komagata & Suzuki, 1987).
Strains: 1, IMCC1021T; 2, M. algae KCTC 12089T; 3, M. mobilis KCTC 12708T; 4, M. phycicola KCTC 22373T; 5, M. ostreae KCTC 23500T. All data were obtained from this study. Fatty acids that represented ,1.0 % are not shown. ECL, equivalent chain-length; tr, trace (,1.0 %); 2, not detected.
The DNA G+C content of strain IMCC1021T was 41.4 mol%, which was within the range of DNA G+C contents reported for the genus Mesonia (32.7–42.1 mol%). The respiratory quinone detected in strain IMCC1021T was menaquinone-6 (MK-6), which is coincident with the major quinone of the family Flavobacteriaceae. The polar lipids found in strain IMCC1021T were phosphatidylethanolamine, two unknown aminolipids and three unknown lipids (Fig. S2). The major cellular fatty acid components (.10 %) of strain IMCC1021T were iso-C15 : 0 (25.1 %), isoC17 : 0 3-OH (18.0 %), C16 : 1v7c and/or C16 : 1v6c (16.6 %) and iso-C15 : 1 G (10.8 %) (Table 2), which were similar to those found in the reference strains of species of the genus Mesonia. Phylogenetic analyses showed that strain IMCC1021T formed a robust clade with species of the genus Mesonia (Fig. 1), as well as sharing similar chemotaxonomic characteristics, including DNA G+C content, quinone, polar lipids and fatty acids. This suggests that strain IMCC1021T can be assigned to this genus. The low 16S rRNA gene sequence similarities (94.9–97.4 %) between IMCC1021T and the available species of the genus Mesonia and the genomic distinctness between strain IMCC1021T and M. mobilis (less than 70 % DNA–DNA hybridization) also suggested that strain IMCC1021T represents a novel genomic species of the genus Mesonia. In addition, phenotypic characteristics, including hydrolysis of macromolecules, enzyme activities and carbon source oxidation (Table 1) differentiated strain IMCC1021T from previously recognized species of the genus Mesonia. Therefore, strain IMCC1021T should be assigned to a novel species of the genus Mesonia, for which the name Mesonia aquimarina sp. nov. is proposed.
Fatty acid (%) Straight chain C15 : 0 C16 : 0 C18 : 0 Branched iso-C15 : 0 iso-C15 : 1 G anteiso-C15 : 0 iso-C16 : 0 iso-C16 : 1 H anteiso-C17 : 1v9c Unsaturated C15 : 1v6c C17 : 1v6c C17 : 1v8c Hydroxy C15 : 0 2-OH C15 : 0 3-OH C17 : 0 2-OH iso-C15 : 0 3-OH iso-C16 : 0 3-OH iso-C17 : 0 3-OH Summed features* 3 (C16 : 1v7c and/or C16 : 1v6c) 9 (C16 : 0 10-methyl and/or iso-C17 : 1v9c) Unknown ECL 13.565
1
2
3
4
5
3.0 1.2 tr
7.3 4.3 4.3
3.6 1.4 2
4.2 tr tr
4.1 2 2
25.1 10.8 2.9 tr 2 2
20.3 7.1 7.5 2.5 1.8 1.4
33.1 9.9 3.7 tr 2 2
31.2 11.7 2.9 1.1 1.5 2
9.7 3.1 16.7 2 2 4.3
tr 1.1 2
2.8 2.4 1.6
1.1 2.0 2
2.7 tr 2
2 3.9 2
1.5 2 1.5 3.8 1.4 18.0
1.7 2 2.3 2.6 3.0 11.7
2 2 1.3 5.5 2.0 16.9
1.3 1.3 1.6 5.1 3.3 12.7
4.2 2 10.4 2 7.7 15.3
16.6
4.2
9.5
9.5
15.7
9.3
6.3
7.1
4.2
4.8
2
2.3
tr
tr
2
*Summed features represent groups of two fatty acids that cannot be separated by the MIDI system.
Gram-staining-negative, oxidase- and catalase-positive, non-motile, non-gliding and aerobic. Cells are rod-shaped
(0.5–0.6 mm61.1–2.0 mm). Colonies are 1–2 mm in diameter, circular, convex, smooth and yellow after 3 days of incubation on MA at 25 uC. Flexirubin-type pigments are not produced. Temperature range for growth is 10–37 uC (optimum, 25 uC). The pH range for growth is pH 5.5–9.5 (optimum, pH 7.0–7.5). Growth occurs at 0.5–15 % (w/v) NaCl [optimum, 3.0–3.5 % (w/v) NaCl]. Gelatin, CMcellulose, casein, Tween 20 and Tween 80 are hydrolysed, but DNA, starch, colloidal chitin and elastin are not. H2S is not produced. Positive for gelatinase activity in API 20NE, but negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, aesculin hydrolysis and b-galactosidase. Positive for the following enzyme activities (API ZYM): alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and b-glucosidase, but negative for lipase (C14), cystine
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International Journal of Systematic and Evolutionary Microbiology 65
Description of Mesonia aquimarina sp. nov. Mesonia aquimarina (a.qui.ma.ri9na. L. fem. n. aqua water; L. adj. marinus from the sea; N.L. fem. adj. aquimarina from seawater).
Mesonia aquimarina sp. nov.
arylamidase, trypsin, a-chymotrypsin, a-galactosidase, bgalactosidase, b-glucuronidase, a-glucosidase, N-acetyl-bglucosaminidase, a-mannosidase and a-fucosidase. Positive for glycerol, erythritol, D-galactose, L-sorbose, L-rhamnose, dulcitol, inositol, D-sorbitol, arbutin, aesculin ferric citrate, melibiose and L-arabitol in API 50CH, negative for Darabinose, L-arabinose, D-ribose, D-xylose, L-xylose, D-adonitol, methyl b-D-xylopyranoside, D-glucose, D-fructose, Dmannose, D-mannitol, methyl a-D-mannopyranoside, methyl a-D-glucopyranoside, N-acetylglucosamine, amygdalin, salicin, cellobiose, maltose, lactose, sucrose, trehalose, inulin, melezitose, raffinose, starch, glycogen, xylitol, gentiobiose, Dturanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, potassium gluconate, potassium 2-ketogluconate and potassium 5-ketogluconate. Susceptible to ampicillin, erythromycin, rifampicin, tetracycline and vancomycin, but resistant to chloramphenicol, gentamicin, kanamycin and nalidixic acid. The respiratory quinone detected is menaquinone 6 (MK-6). The major phospholipids are phosphatidylethanolamine, aminophosphoslipids and unknown polar lipids. The major cellular fatty acids are iso-C15 : 0, iso-C17 : 0 3-OH, C16 : 1v7c and/or C16 : 1v6c and iso-C15 : 1 G. The type strain of the species is strain IMCC1021T (5KCTC 32320T5NBRC 109485T), isolated off the coast of the East Sea, Korea. The G+C content of the genomic DNA of the type strain is 41.4 mol%.
Acknowledgements This study was supported by the project on survey of indigenous species of Korea of the National Institute of Biological Resources (NIBR), Ministry of Environment (Korea), by Mid-Career Research Program through NRF funded by the Ministry of Science, ICT and Future Planning (NRF-2013R1A2A2A01068004), and by the Inha University Research Grant.
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